The microfluidic system consists of ten microfluidic chips of micro-ellipse filters, plastic tubes, syringes and syringe pumps. A syringe pump was used to load cell suspension into the chip. The syringe attached to the pump was connected to inlet of the chip through plastic tubing as shown in Fig. 3 . An inverted phase contrast fluorescence microscope (Nikon, Japan) equipped with a high speed CCD camera (Nikon, Japan) was used to observe.
Inverted phase contrast fluorescence microscope
Manufactured by Nikon
Sourced in Japan
The Inverted Phase Contrast Fluorescence Microscope is a laboratory instrument designed for high-resolution imaging of transparent and fluorescent samples. It combines phase contrast and fluorescence microscopy techniques to provide enhanced contrast and visualization of cellular structures and processes. The core function of this microscope is to enable detailed observation and analysis of specimens, such as living cells, tissues, and microorganisms, through the integration of these complementary imaging modalities.
Automatically generated - may contain errors
Lab products found in correlation
High speed ccd camera, by Nikon (3 mentions)
Calcein am, by Biotium (1 mentions)
Hoechst, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dihydroethidium dhe assay kit, by Beyotime (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Rabbit anti map2 monoclonal antibody, by Abcam (1 mentions)
Triton x 100, by Merck Group (1 mentions)
Alexa fluor 647 conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
8 protocols using inverted phase contrast fluorescence microscope
1
Microfluidic Chip Cell Filtration
2
Spiral Microfluidic Chip for Cell Separation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The microfluidic system is composed of the triplet-microchannel spiral microfluidic chip (Spiral-Slits Chip), plastic tubing, syringes and syringe pumps17 (link). A syringe pump was used to load cell suspension into Spiral chip. The syringe attached to the pump was connected to the inlet of the triplet-microchannel spiral microfluidic chip through plastic tubing30 . An inverted phase contrast fluorescence microscope (Nikon, Japan) equipped with a high-speed CCD camera (Nikon, Japan) was used to observe. This device was fabricated in Polydimethylsiloxane (PDMS) bonded to a glass substrate17 (link).
3
Culturing and Characterizing Cancerous Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MCF-7 cells (human breast adenocarcinoma) and HepG2 cells (hepatocellular carcinoma) were provided by Tianjin Medical University Cancer Institute and Hospital. Hela cells were offered by Peking University Third Hospital. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone, USA) medium supplemented with 10% fetal bovine serum (FBS) (GIBCO, USA) and 1% penicillin-streptomycin (Ying Reliable biotechnology, China) and incubated in a humidified atmosphere at 37 °C with 5% CO atmosphere. When cell lines were grown as adherent monolayers to 95% confluence, they were detached from the culture dishes with 0.25% Trypsin solution. The cell suspension was diluted to obtain a desired cell concentration using a hemocytometer. Cells were labeled by Calcein AM (BIOTIUM, USA) and Hoechst (Molecular Probes, Solarbio Corp., China). Observation of CTCs was used an inverted phase contrast fluorescence microscope (Nikon, Japan) equipped with a high speed CCD camera (Nikon, Japan) and counted by using hemocytometer to around 100 cells in 1 ml PBS containing 1% BSA and 0.05% tween-20.
4
Quantifying Cellular Oxidative Stress and Autophagy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reactive oxygen species (ROS) production was detected by an ROS-sensitive fluorescent indicator using dihydroethidium (DHE) assay kit (Beyotime Biotechnology, Shanghai, China). Briefly, the H9C2 cells were seeded in 24-well plates (5 × 104 cells/well) with 2 μM DHE solution at 37 °C for 30 min, and then the cells were washed for 3 times with PBS. ROS generation was represented by the total DHE fluorescence, and fluorescence intensity was observed and imaged with the inverted fluorescence phase contrast microscope (Nikon Corporation, Tokyo, Japan). The fluorescence intensity was analyzed using Image J software (NIH, Bethesda, MD).
GFP-LC3 plasmid was used to infect H9C2 cells for autophagy detection.
In brief, the H9C2 cells were inoculated into a 24-well plate (5 × 104 cells/well), then plasmid containing GFP-LC3B was transfected into the cells with lipofectamine 3000 for 48 h according to the manufacturer's protocol. Images of cells were captured under a fluorescence phase contrast microscope (Nikon Corporation, Tokyo, Japan).
GFP-LC3 plasmid was used to infect H9C2 cells for autophagy detection.
In brief, the H9C2 cells were inoculated into a 24-well plate (5 × 104 cells/well), then plasmid containing GFP-LC3B was transfected into the cells with lipofectamine 3000 for 48 h according to the manufacturer's protocol. Images of cells were captured under a fluorescence phase contrast microscope (Nikon Corporation, Tokyo, Japan).
5
Organoid formation from arsenic-treated cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
CellTracker Green CMFDA-labeling C3H10T 1/2 or MC3T3-E1 cells following the manufacturer's instructions were treated with different arsenic formulations (5 or 10 μmol/L) for 24 h. After removing the medium, pretreated C3H10T 1/2 or MC3T3-E1 cells were seeded into 96 well ultra-low attachment plates (20,000 cells/well) and cultured with untreated 4T1-RFP cells (10,000 cells/well). The heterotypic organoids were grown for 4 days. The morphology and fluorescence image of organoids were captured with inverted fluorescence phase contrast microscope (Nikon). The sizes of organoids were determined by Image J software (National Institutes of Health).
6
Establishing Stable Cell Lines with shRNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The LLC-PK1 cells were seeded (2 × 104/well) into 6-well plates and incubated for 24 h at 37 °C in a 5% CO2 atmosphere. When reaching 50–70% confluence, the cells were washed three times with sterilized 0.01 M pH7.4 phosphate buffered saline (PBS). Afterwards, the cells were transfected with 2.5 μg/well of shRNA-expressing plasmids using a LipofectamineTM 3000 (Invitrogen, Waltham, MA, USA), according to the manufacturer’s instructions. After 24 h of incubation, the growth media were substituted with maintenance media containing 2% FBS and 1000 μg/mL of Neomycin (G418). The survival cell clones were maintained in G418-containing media for 15 d with frequent media replacements until cell death could no longer be observed. Then, these monoclonal cells transfected with shRNA-expressing plasmids were screened by limiting dilution analysis (LDA), as previously described [29 (link)], and cultured in DMEM growth media containing G418 (500 μg/mL) in 6-well plates at 37 °C in a 5% CO2 atmosphere. After reaching a confluence, these plasmid-transduced cells were inoculated with PDCoV at a multiplicity of infection (MOI) of 0.1. Non-transfected cells were set as a control. Cell transfection efficiency and cytopathic effect (CPE) images were taken under an inverted fluorescence/phase-contrast microscope (Nikon, Tokyo, Japan).
7
Immunocytochemical Characterization of NSCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NSCs were identified using standard immunocytochemical staining for Nestin, while differentiated NSCs were identified by staining for GFAP, GALC, NF-H and MAP2, which are markers of astrocytes, oligodendrocytes and neurons, respectively. Cells were fixed with paraformaldehyde (4%, w/v) in PBS and then permeabilized with 0.1% Triton X-100 (Sigma) at room temperature for 10 min. After being blocked with 5% BSA for 1 h, the cells were washed and incubated with primary rabbit anti-Nestin polyclonal antibody (1:500 dilution; Bioss, Beijing, China), primary rabbit anti-GFAP polyclonal antibody (1:200 dilution; Bioss), primary rabbit anti-GALC polyclonal antibody (1:200 dilution; Absin, Shanghai, China), primary rabbit anti-NF-H polyclonal antibody (1:200 dilution; Bioss), and rabbit anti-MAP2 monoclonal antibody (1:500 dilution; Abcam) at 4°C overnight. After three washes, the cells were incubated with a goat anti-rabbit IgG-Cy3 secondary antibody (1:200 dilution; Absin) at room temperature for 2 h. The nuclei were counterstained with DAPI at room temperature for 10 min. The samples were visualized with a fluorescence phase contrast inverted microscope (Nikon, Tokyo, Japan).
8
Immunohistochemical Analysis of Chl1 in Mouse Embryo Brains
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Immunohistochemical analyses were performed on six paraffinembedded mouse embryo brain tissues collected from six independent dams at E10.5 to detect the localization of Chl1-positive cells. Briefly, sections were treated with 3% hydrogen peroxide for 20 min to block endogenous peroxidase activity. For antigen retrieval, the slides were placed in racks containing EDTA buffer (pH 9.0) and heated in a microwave oven at maximum power for 10 min. After that, the sections were incubated with primary antibodies against Chl1 (1:100 dilution; R&D) at 4°C overnight. Then, the slides were incubated with an Alexa Fluor 647-conjugated secondary antibody (1:500 dilution; Invitrogen) at room temperature for 1 h. The sections were counterstained with DAPI to identify the nuclei and then mounted with Permount. Photomicrograph images were captured using a fluorescence phase contrast inverted microscope (Nikon) with Picture Frame software.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!